Dianthus cruentus Griseb. (Caryophyllaceae) is a herbaceous perennial species native to the Balkan Peninsula, including Greece, and western Asia. It is a species of great ornamental interest due to its architectural inflorescences and its compact blue-green foliage, particularly resistant to dry and poor soils and attractive to pollinators such as butterflies and bees. In addition, it is a species of medical potential due to its strong antioxidant action.
In the present study, the determination of the appropriate conditions for maximizing the germination of its seeds in both in vitro and in vivo conditions was initially investigated. Two seed lots collected from a native population of the species on Mount Kallidromo, Fthiotida, dry-stored for 0 or 12 months, were used, without any pre-treatment.
Regarding in vitro germination, the seeds were surface sterilized with a 20% commercial bleach water solution for 10 minutes, were then rinsed with distilled water and afterwards were placed in Petri dishes containing ½ MS medium, under 16 h photoperiod from 37.5 μmol m-2 s-1 fluorescent light, at temperatures of 5, 10, 15, 20 or 25 °C. The optimum temperature for seed germination was 15 °C, in which the highest germination ability in both recently collected (98%) and 12-month-old seeds (100 %) was observed. The lowest germination percentages were recorded at the two extreme temperatures of 5 °C and 25 °C, with particularly low values for recently collected seeds (18% and 15%). At all temperatures, minus that of 5 °C, the seeds germinated rapidly (T50 = 2-6 d). The duration of the seeds’ dry storage significantly affected their germination, increasing the germination percentage at the 2 extreme temperatures and demonstrating the positive effect of dry afterripening. As for ex vitro germination, the seeds were placed in pots with a mixture of peat-perlite (1: 1, v/v) and were incubated in growing chambers at temperatures of 15, 20 or 25 °C. The highest germination percentage was observed at 15 °C for both seed lots (97-99%), while the lowest at the temperature of 25 °C (62% and 4% for the 0- and 12-months-old seeds respectively). Germination ability was slightly lower in ex vitro germination, especially at 25 °C.
Furthermore, the micropropagation of the species from explants excised from 2-month-old seedlings was investigated on solid substrates. Substrates containing MS medium and 8 g L-1 agar were used in almost all substages of the proliferation stage, and the effects of cytokinins BA, 2iP and ZEA, with the absence or the presence of auxin NAA, were investigated, using explants derived from non-hyperhydric or hyperhydric shoots. In the initial culture substrates containing 0.1 mg L-1 BA or 0.1 or 0.5 mg L-1 2iP the explants produced shoots at a high percentage (72-87%), but many of these were shown to be hyperhydric, with abnormal morphology and vitreous appearance. The number of hyperhydric shoots and the number of nodes seemed to be higher on substrates containing 2iP, with the largest number of normal shoots (2.3) appearing in the substrate containing 0.1 mg L-1 BA.
During the 1st subculture stage, hyperhydric shoots were observed in bigger numbers in the substrate containing 2iP, while an increase in shoot proliferation and the number of hyperhydric shoots was correlated with the use of hyperhydric explants grown on hormone-free MS substrate. In the 2nd subculture stage, the presence of 0.1 mg L-1 BA yielded the largest number of shoots regardless of the physiology of the explants, while the addition of 0.05 mg L-1 resulted in a significant increase in the number of normal shoots and a reduction in hyperhydricity rates when hyperhydric explants were used. In the 3rd subculture, when explants derived from nonhyperhydric shoots were used the highest value of the multiplication index (4.5) and the maximum number of shoots (3.6) was recorded in MS substrate containing 2 mg L-1 BA. Increasing the concentration of agar to 12 g L-1 produced the largest number of shoots (2.4) when hyperhydric explants were used. In both cases, the highest percentages of hyperhydricity were recorded on MS substrate containing 0.1 mg L-1 ZEA. In contrast, during the 4th subculture, the use of this substrate resulted in the greatest multiplication index value at this stage (4.8).
Overall, the use of MS substrate with 0.1 mg L-1 BA and 0.05 mg L-1 NAA resulted in the highest values of the proliferation index (5.1) regardless of the physiology of the explants used, with its maximum value (5.2) recorded from the culture of hyperhydric explants on MS substrate containing 0.1 mg L-1 BA and 12 g L-1 agar. The presence of phytohormones in the substrates increased the production of callus around the base of the explants, while high concentrations of cytokinins lessened the occurence of roots.
D. cruentus microshoots were rooted in MS or ½ MS substrates in the presence or absence of IBA. The use of MS substrate resulted in lower rooting rates (55-70%) when lower concentrations of IBA were used (0 or 0.1 mg L-1). The optimal concentration of IBA was found to be 0.5 mg L-1 regardless of the multiplication substrate where the microshoots originated or the nutrient medium used in the rooting substrate, with rooting rates of 83-100%, while in some instances a carry over effect, caused by the cytokinin content of the shoot proliferation substrates, was observed on the roots’ number and median length.
The ex vitro acclimatization of the rooted microshoots was completed with a success rate of 91% using a peat:perlite substrate (1:1, v/v). Significant morphological differences were observed between acclimatized plantlets, including differences in stem length and total number of shoots and nodes, which were correlated with the influence of different shoot proliferation and/or rooting substrates, while the presence of IBA during rooting seemed to have an amendable effect on their survival. Finally, the use of an integrated, 60-days-long propagation protocol with shoot proliferation and rooting occurring concurrently in one stage on MS substrates free of PGRs or containing BA or 2iP and NAA produced rooted young plants at a high percentage (88%), which had the maximum observed total number of shoots and nodes and were then successfully acclimatized, surviving at a percentage of 86%.
Scientific area: Horticulture
Keywords: Dianthus cruentus, native ornamental plant, germination ecophysiology, seed germination in vitro, seed germination temperature, seed lot age, dry afterripening, in vitro propagation, solid culture, seedling explant, hyperhydricity, hyperhydric explant, callus induction, in vitro rooting, ex vitro acclimatization, cytokinins, IBA, carry over effect, acclimatized plantlets’ morphology